The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase

Eur J Biochem. 2003 Mar;270(6):1301-7. doi: 10.1046/j.1432-1033.2003.03500.x.

Abstract

Site-directed mutagenesis was used to investigate the control of 2-oxoacid cosubstrate selectivity by deacetoxycephalosporin C synthase. The wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids (e.g. 2-oxohexanoic acid, 2-oxo-4-methyl-pentanoic acid) as the cosubstrate. The following mutant enzymes were produced: R258A, R258L, R258F, R258H and R258K. All of the mutants have broadened cosubstrate selectivity and were able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization by all mutants was decreased as compared to the wild-type enzyme, and in some cases activity was abolished with the natural cosubstrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ampicillin / metabolism
  • Arginine / metabolism*
  • Intramolecular Transferases / genetics
  • Intramolecular Transferases / metabolism*
  • Ketoglutaric Acids / metabolism
  • Models, Molecular
  • Molecular Structure
  • Mutagenesis, Site-Directed*
  • Oxidation-Reduction
  • Penicillin-Binding Proteins*
  • Penicillins / metabolism
  • Substrate Specificity

Substances

  • Ketoglutaric Acids
  • Penicillin-Binding Proteins
  • Penicillins
  • Ampicillin
  • Arginine
  • Intramolecular Transferases
  • deacetoxycephalosporin C synthetase